Glycosylation

Glycoprofile I

Product Code PP0200

Removal of the carbohydrate groups on a glycoprotein is recommended prior to protein identification. In general, glycoproteins and glycopeptides do not ionize completely during MS analysis, leading to inadequate spectral data. Glycopeptides have lower detection sensitivity due to microheterogeneity of the attached glycans, resulting in signal suppression in MS analysis. Thus, removing the glycans increases protein sequence coverage for protein identification. Furthermore, proteolytic cleavage is a prerequisite for the elution of peptide fragments out of gels and identification by MS. However, proteolytic digestion of the native glycoprotein is often incomplete due to steric hindrance from the bulky oligosaccharides present on the molecule. Information gained from MS analysis of deglycosylated tryptic peptides can be used to locate glycosylation sites on the glycoprotein.



The GlycoProfile™ In-Gel Enzymatic N-Deglycosylation Kit is optimized to provide a convenient and reproducible method to N-deglycosylate and digest protein samples from 1D or 2D polyacrylamide gel pieces for subsequent MS or HPLC analysis. The procedure is suitable for Coomassie® Brilliant Blue and colloidal Coomassie stained gels. Silver stained gels may also be used if properly destained.



The kit includes PNGase F and trypsin enzymes necessary for N-linked deglycosylation and tryptic digestion, respectively. The samples can then be desalted and concentrated for analysis by MALDI-TOF MS or Electrospray MS with subsequent database searching.

Proteomics Grade PNGase F is extensively purified and lyophilized from potassium phosphate buffer to produce a stable product. The material is free from glycerol and other stabilizers, and contains very low levels of buffer salts. This enzyme gives excellent performance when used for in-gel N-linked deglycosylation of glycoproteins and glycopeptides. PNGase F releases asparagine-linked oligosaccharides from glycoproteins and glycopeptides by hydrolyzing the amide of the asparagine (Asn) side chain.

The GlycoProfile In-Gel Enzymatic N-Deglycosylation Kit provides adequate reagents to deglycosylate and digest a minimum of 10 samples according to the procedure described here using yeast invertase as a model glycoprotein.

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GlycoProfile™ I: In-Gel Enzymatic N-Deglycosylation Kit

 

The GlycoProfile In-Gel Enzymatic N-Deglycosylation Kit is composed of 8 reagents:
Destaining Solution - Solution to remove dye bound to the protein of interest.
Proteomics Grade PNGase F
Proteomics Grade Trypsin
Trypsin Reaction Buffer
Invertase - A control glycoprotein standard
Peptide Extraction Solution
Biotech Grade Acetonitrile

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